Protein Engineering Protocols - Mycobacteriology research center

Ribosome-Inactivation Display System 2333.2.4. Polymerase Chain ReactionPerform PCR using thermostable DNA polymerase. In our model study, PCRwas performed by KOD Dash ® (Toyobo) according to the supplier’s recommendation.1. Mix 20 µL cDNA, 10 µL of 10X PCR buffer, 10 µL of each 2 mM dNTP, 1 µL of10 µM RT primer (as the down primer), and 2 µL of 10 µM (as the up primer).Adjust the volume of the solution to 99 µL with distilled water, without addingKOD Dash. The up primer (5′-AAT TTT GTT TAA CTT TAA GAA GGA G-3′)that recognizes the downstream portion of the T7 promoter sequence was used.2. Denature the mixture at 98°C for 2 min and chill on ice immediately.3. Add 0.5 µL of KOD Dash.4. Perform PCR cycles (1 min at 98°C, followed by 15 to 30 cycles of 10 s at 98°C,2 s at 55°C, and 30 s at 72°C; and finished by 10 min at 72°C). Incubate at 98°Cfor 1 min.4. Notes1. The Flexi ® Rabbit Reticulocyte Lysate system provides flexibility of reaction conditionsin comparison with standard rabbit reticulocyte lysate systems. The reducingagents, including dithiothreitol, should be omitted for the display of proteinscontaining disulfide bridges. The concentrations of Mg 2+ and K + ions were optimizedby referring to the ribosome display method (32).2. We occasionally detected a high background in the affinity selection of GST by glutathionesepharose. However, we did not detect background in the affinity selectionof GST by antibody against GST instead of glutathione sepharose. The result mayshow that several GST displayed on ribosomes cannot dimerize because of sterichindrance, because only GST dimers recognize glutathione.3. The ribosomal complexes are stabilized by adding 5 to 50 mM of the MgCl 2.4. Although we succeeded in the selection of GST or streptavidin from the pool of themixture of GST and streptavidin at room temperature, we occasionally detected nonspecificbinding. It seems that the ribosomal complexes are stabilized by chilling thetranslation mixture. Surprisingly, by adding MgCl 2to the binding buffer, and chillingthe translation mixture, a small amount of StAv-mR mRNA can bind to biotin agarosedespite the presence of a stop codon. It seems that an mRNA–ribosome–protein complexis formed in the ribosome display system with a stop codon.AcknowledgmentsThe authors thank Prof. J. D. Robertus of the University of Texas for the kindgift of plasmid pUTA, and Prof. M. Sisido and Dr. T. Hosaka of the University ofOkayama for plasmid pGSH. The authors also thank members of the Taira laboratory,especially Dr. Loura Nelson, for helpful comments and discussions. Thisresearch was supported by grants from Promotion of Basic Research Activities for